Eukaryotic cells transiently halt cell cycle progression is the presence of damaged or incompletely replicated genomic DNA to allow for repair or completion of replication before mitotic chromosomal segregation. The mechanisms that control cell cycle progression following genotoxin exposure or replication arrest are called the DNA damage and replication checkpoints, respectively. The disruption of these fail-safe mechanisms leads to genomic instability and, ultimately, cancer. Rad17, Rad9, Hus1, and Rad1 are essential for checkpoint activation. Sequence alignments indicate that Rad17 shares homology with the RFC subunits. Moreover, Rad17 interacts with the small RFC subunits into an independent complex (Rad17-RFC). Rad1, Hus1, and Rad9 assemble into a complex called the 9-1-1 complex that is structurally related to PCNA. The 9-1-1 complex interacts with Radl7. A model has been proposed in which Radl7-RFC acts as a DNA structure-specific sensor, clamping 9-1-1 complex at sites of damage and activating checkpoint signaling. The Specific Aims of this proposal are to: 1) dissect the role of Rad17's ATPase domain in genotoxic- and replication arrest-induced checkpoint signaling initiation and cell cycle arrest and 2) determine the function of Rad17 phosphorylation in 9-1-1 chromatin binding and checkpoint activation following genotoxin exposure or chromosomal replication arrest. For this purpose, we will reconstitute XRad17-depleted Xenopus laevis cytoplasmic egg extracts with insect cell-expressed wild-type and mutant Rad17-RFC. Collectively, these studies will biochemically dissect the role of Rad17 in DNA damage and replication arrest checkpoint signaling pathway of higher eukaryotes.